The interferon γ pathway enhances pluripotency and X-chromosome reactivation in iPSC reprogramming

Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) requires activation of the pluripotency network and resetting of the epigenome by erasing the epigenetic memory of the somatic state. In female mouse cells, a critical epigenetic reprogramming step is the reactivation of the inactive X chromosome. Despite its importance, a systematic understanding of the regulatory networks linking pluripotency and X-reactivation is missing. Here, we reveal important pathways for pluripotency acquisition and X-reactivation using a genome-wide CRISPR screen during neural precursor to iPSC reprogramming. In particular, we discover that activation of the interferon γ (IFNγ) pathway early during reprogramming accelerates pluripotency acquisition and X-reactivation. IFNγ stimulates STAT3 signaling and the pluripotency network and leads to enhanced TET-mediated DNA demethylation, which consequently boosts X-reactivation. We therefore gain a mechanistic understanding of the role of IFNγ in reprogramming and X-reactivation and provide a comprehensive resource of the molecular networks involved in these processes.

Flow cytometry analysis during 6 days of doxycycline treatment in the X-GFP iCas9 ESC line was done to measure the X-GFP percentage decay in cells containing a gRNA targeting the GFP gene.Gating shows the X-GFP+ population.(B) Percentage of gRNA representation in the plasmid library, infected ESCs and the 4 populations analyzed in two independent screening rounds: NPCs and day 10 reprogramming populations (non-pluripotent, early pluripotent, late pluripotent).Error bars represent SD. (C) gRNA abundance comparisons (related to D-I): NPCs to non-pluripotent, early pluripotent and late pluripotent populations.(D) Pathways related to common underrepresented genes (n=927 genes) in the three reprogramming populations compared to NPCs (WikiPathways Mouse 2019).For all comparisons, an RRA score < 0.05 and Log2FC < -0.75 (underrepresented) filtering was applied.(E-G) Representation of genes with negative Log2FC (underrepresented) vs -log10 RRA in the non-pluripotent (E), early pluripotent (F) and late pluripotent (G) populations compared to NPCs (RRA cutoff = 0.05, Log2FC cutoff = -0.75).(H) Venn diagram (using Venny 2.1.0)representing overlap of underrepresented genes (compared to NPCs) in each of the sorted populations at day 10 of reprogramming.(I) Bar plot showing percentages of common and unique underrepresented genes (compared to NPCs) in each of the sorted populations at day 10 of reprogramming.(J) Pathways (WikiPathways Mouse 2019) related to underrepresented genes in the "early pluripotent vs non-pluripotent" comparison (activators of early pluripotency, n=1361 genes) (RRA score < 0.05 and Log2FC < -0.8 filtering was applied).(K) Pathways (WikiPathways Mouse 2019) related to overrepresented genes in the "early pluripotent vs non-pluripotent" comparison (repressors of early pluripotency, n=693 genes) (RRA score < 0.05 and Log2FC > 0.8 filtering was applied).(L) Representation of genes with positive Log2FC (overrepresented) vs -log10 RRA (RRA cutoff = 0.05, Log2FC cutoff = 0.75) in the "early pluripotent vs non-pluripotent" comparison (repressors of early pluripotency).(M) Representation of genes with positive Log2FC (overrepresented) vs -log10 RRA (RRA cutoff = 0.05, Log2FC cutoff = 0.75) in the "late pluripotent vs early pluripotent" comparison (repressors of late pluripotency, X-reactivation).(N) Pathways (WikiPathways Mouse 2019) related to overrepresented genes in the "late pluripotent vs early pluripotent" comparison (repressors of late pluripotency, X-reactivation, n=839 genes) (RRA score < 0.05 and Log2FC > 0.8 filtering was applied).The number (n) of counted colonies is indicated in the graph.NANOG+ or X-GFP+ colonies were scored as low or high if approximately less or more than half of the cells in the colony were positive for these markers, respectively.(F) Immunofluorescence (high magnification, 63x) for NANOG and X-GFP (active X chromosome) of day 7 reprogramming colonies +/-IFNγ treatment.Scale bar = 50 µm.(G) Percentages of NANOG+ and X-GFP+ (from NANOG+) cells from immunofluorescence in (F).The number (n) of counted cells is indicated in the graph.Δβ-values for 5mC in day 7 X-GFP-negative iPSCs for each genomic region in autosomes and X chromosome (corresponding to analysis in (C)).Bars marked with "ns" correspond to non-significant changes from analysis in (C).(E) X-chromosome paint DNA FISH in control X-GFP-negative and IFNγ-treated (d0-5) X-GFP-negative and -positive day 7 iPSCs (scale bar = 5 µm) and quantification of cells with 1 or 2 X chromosomes.B, D) Δβ-values for 5mC in day 5 (B, corresponding to analysis in A) or 5hmC in day 7 X-GFP+ iPSCs (D, corresponding to analysis in C) for each genomic region in autosomes and X chromosomes.Bars marked with "ns" correspond to non-significant changes from analysis in (A) or (C).(E) Transcription factor binding site (TFBS) enrichment analysis on differentially methylated Xchromosomal CpGs (DMPs, logFC<(-0.1),p<0.01, n=468 CpGs) which lose methylation upon IFNγ treatment compared to control in day 7 X-GFP+ iPSCs.-log10(FDR) capped values are above 25.(F) Analysis of 5mC and 5hmC levels (β-values) of CpGs in early and main X-reactivating gene promoters at day 5 and day 7 X-GFP+ iPSCs for control and IFNγ conditions (gene lists were obtained from ( 24   Allele-specific analysis of 5mC and 5hmC percentages (relative to total C) at day 5 of reprogramming in control and IFNγ-treated samples, in specific promoter loci surrounding CpGs from X-reactivating genes that were found as differentially hydroxymethylated on day 5 or 7 in Fig. 5, including escapee gene controls.Analysed loci contained promoter regions of the escapee genes Ddx3x (2 CpGs) and Eif2s3x (16 CpGs) (plotted together because of low variability), and the X-reactivating genes Mtm1

Supplemental Tables Description
Supplemental Table S1.MAGeGK gene summary for CRISPR screen comparisons.Related to Fig. 1 and fig.S1.Statistical comparisons for each gene in "non-pluripotent vs NPCs", "early pluripotent vs NPCs", "late pluripotent vs NPCs", "early vs non-pluripotent" and "late vs early pluripotent".S2.Lists of genes and pathways for CRISPR screen comparisons.Related to Fig. 1 and fig.S1.Gene lists for each category and pathways ("WikiPathways mouse 2019") corresponding to each of them: essentialome, repressors of colony formation, drivers and repressors of early pluripotency, drivers and repressors of late pluripotency and X-reactivation.S3.DESeq2 and pathway analysis from RNA-sequencing experiments.Related to Fig. 3 and fig.S5.Differential gene expression analysis for each reprogramming timepoint (IFNγ vs control) and pathways ("WikiPathways mouse 2019") associated to them (day 2, day 5, day 7 X-GFP negative, day 7 X-GFP medium, day 7 X-GFP high), and allelic ratio for X-linked genes in ESCs, NPCs and each reprogramming population.S4.DNA methylation: DMPs, TFBS enrichment, gene lists and pathway analysis.Related to Fig. 5, fig.S8 and fig.S9.Differentially methylated CpGs (DMPs) for 5mC at days 5 and day 7 (X-GFP+) iPSCs (IFNγ vs control); overlap of upregulated genes in day 7 X-GFP+ cells by RNA-seq and genes with lower promoter 5mC levels in day 7 X-GFP+ cells and pathways (WikiPathways mouse 2019) associated to them; lists of X-reactivating, "early" and "main" X-reactivating, and escapee genes obtained from (24), SeSAMe TFBS enrichment (based on ChIP-seq data from Cistrome/ENCODE databases) at days 5 and 7 for CpGs losing 5mC globally, and for CpGs losing 5mC on the X chromosome at day 7. S5.Resources: oligonucleotides, antibodies, molecules for pathway validation, cell lines and softwares used in this study.S6.Source data for figure panels.

Fig. S1 .
Fig. S1.CRISPR screen reveals molecular networks involved in reprogramming and X-chromosome reactivation.Related to Fig. 1. (A) Validation of knockout efficiency by flow cytometry.Flow cytometry analysis during 6 days of doxycycline treatment in the X-GFP iCas9 ESC line was done to measure the X-GFP percentage decay in cells containing a gRNA targeting the GFP gene.Gating shows the X-GFP+ population.(B) Percentage of gRNA representation in the plasmid library, infected ESCs and the 4 populations analyzed in two independent screening rounds: NPCs and day 10 reprogramming populations (non-pluripotent, early pluripotent, late pluripotent).Error bars represent SD. (C) gRNA abundance comparisons (related to D-I): NPCs to non-pluripotent, early pluripotent and late pluripotent populations.(D)Pathways related to common underrepresented genes (n=927 genes) in the three reprogramming populations compared to NPCs (WikiPathways Mouse 2019).For all comparisons, an RRA score < 0.05 and Log2FC < -0.75 (underrepresented) filtering was applied.(E-G) Representation of genes with negative Log2FC (underrepresented) vs -log10 RRA in the non-pluripotent (E), early pluripotent (F) and late pluripotent (G) populations compared to NPCs (RRA cutoff = 0.05, Log2FC cutoff = -0.75).(H) Venn diagram (using Venny 2.1.0)representing overlap of underrepresented genes (compared to NPCs) in each of the sorted populations at day 10 of reprogramming.(I) Bar plot showing percentages of common and unique underrepresented genes (compared to NPCs) in each of the sorted populations at day 10 of reprogramming.(J) Pathways (WikiPathways Mouse 2019) related to underrepresented genes in the "early pluripotent vs non-pluripotent" comparison (activators of early pluripotency, n=1361 genes) (RRA score < 0.05 and Log2FC < -0.8 filtering was applied).(K) Pathways (WikiPathways Mouse 2019) related to overrepresented genes in the "early pluripotent vs non-pluripotent" comparison (repressors of early pluripotency, n=693 genes) (RRA score < 0.05 and Log2FC > 0.8 filtering was applied).(L) Representation of genes with positive Log2FC (overrepresented) vs -log10 RRA (RRA cutoff = 0.05, Log2FC cutoff = 0.75) in the "early pluripotent vs non-pluripotent" comparison (repressors of early pluripotency).(M) Representation of genes with positive Log2FC (overrepresented) vs -log10 RRA (RRA cutoff = 0.05, Log2FC cutoff = 0.75) in the "late pluripotent vs early pluripotent" comparison (repressors of late pluripotency, X-reactivation).(N) Pathways (WikiPathways Mouse 2019) related to overrepresented genes in the "late pluripotent vs early pluripotent" comparison (repressors of late pluripotency, X-reactivation, n=839 genes) (RRA score < 0.05 and Log2FC > 0.8 filtering was applied).

Fig. S5 .
Fig. S5.Transcriptomic analysis of interferon γ pathway activation during iPSC reprogramming.Related to Fig. 3. (A) Flow cytometry plots of X-GFP expression (from SSEA1+ cells) in control and IFNγtreated day 7 iPSCs.Gating shows sorted populations for RNA-sequencing.Average percentages between two independent reprogramming inductions are indicated for each population.(B, C) Principal component analysis of RNA-sequencing of NPCs, day 2, day 5, day 7 reprogramming populations and ESCs, in control and IFNγ treatment (day 0-5), representing the top 500 most variable autosomal genes only (B) and Xchromosomal genes only (C).(D) Expression (FPKM) of selected genes (Stat1, Nanog, Prdm14 and Esrrb) in NPCs, ESCs, day 2, day 5 and day 7 reprogramming populations +/-IFNγ treatment (two RNAsequencing replicates shown).(E) Venn diagram (using Venny 2.1.0)representing overlapping of upregulated and downregulated genes upon IFNγ treatment between day 2 dox-treated cells and day 7 X-GFP medium cells.(F) MA plot displaying transcriptomic changes of IFNγ vs control day 5 iPSCs (adjusted p value = 0.1).Upregulated genes are highlighted in light blue, downregulated genes are highlighted in orange.Selected genes are shown with points in red.(G, H) Upregulated (G) and downregulated (H) pathways in IFNγ vs control day 5 iPSCs (WikiPathways Mouse 2019) (adjusted p value = 0.1).(I) MA plot displaying transcriptomic changes of IFNγ vs control day 7 X-GFP negative iPSCs (adjusted p value threshold = 0.1).Upregulated genes are highlighted in light blue, downregulated genes are highlighted in orange.Selected genes are shown with points in red.(J, K) Upregulated (J) and downregulated (K) pathways in IFNγ vs control day 7 X-GFP negative iPSCs (WikiPathways Mouse 2019) (adjusted p value = 0.1).

Fig. S8 .
Fig. S8.Day 7 X-GFP-reprogramming cells show a reduction in Xist clouds and in H3K27me3 spots, and equal X-chromosomal DNA methylation levels.Related to Fig. 4. (A) RNA FISH showing Xist clouds on the inactive X chromosome (Sx9 probe) in NPCs and day 7 SSEA1+ X-GFP-negative/positive control/IFNγ iPSCs (scale bar = 5 µm) and quantification of Xist cloud-positive and -negative cells in all conditions.(B) H3K27me3 immunofluorescence in NPCs and day 7 SSEA1+ X-GFP negative/positive control/IFNγ iPSCs (scale bar = 5 µm) and quantification of H3K27me3 spot-positive and -negative cells in all conditions.(C) Analysis of 5mC levels (β-values) of CpGs on autosomes and X chromosome in day 7 X-GFP-negative iPSCs for control and IFNγ conditions, globally and divided by genomic distribution: promoters (<= 1kb from TSS), gene bodies and distal regions (number (n) of detected CpGs from each category is indicated on the bottom of the graphs).Δβ-values (mean β-value IFNγ -mean β-value control) and p values (comparison IFNγ vs control) are shown in the graphs.Statistics (unpaired t-tests): ns = nonsignificant; * = p<0.05;**** = p<0.0001.(D)Δβ-values for 5mC in day 7 X-GFP-negative iPSCs for each genomic region in autosomes and X chromosome (corresponding to analysis in (C)).Bars marked with "ns" correspond to non-significant changes from analysis in (C).(E) X-chromosome paint DNA FISH in control X-GFP-negative and IFNγ-treated (d0-5) X-GFP-negative and -positive day 7 iPSCs (scale bar = 5 µm) and quantification of cells with 1 or 2 X chromosomes.

Fig. S11 .
Fig. S11.Absence of TET1 does not impede IFNγ-mediated enhanced X-GFP reactivation at day 7 of reprogramming.Related to Fig. 5. (A) PCR on genomic DNA of parental (WT) and Tet1-/-clones showing an around 200 bp deletion in exon 3. The asterisk marks the clones used for further experiments.(B) Schematic representation of Sanger sequencing (and amino acid equivalence) of PCR products from genomic DNA in the parental clone (WT) and 5 Tet1-/-clones used for the experiment, which showed a premature STOP codon (represented in black).(C) Experimental design for (D): Tet1-/-, parental and scrambled gRNA control ESCs were differentiated into NPCs and then reprogrammed into iPSCs in the presence or absence of IFNγ (day 0-5).X-GFP percentages (from SSEA1+ cells) were measured by flow cytometry at day 7 of reprogramming. 3 clones from the parental cell line, 3 clones containing a scrambled gRNA and 5 Tet1 -/-clones were used, including three technical replicates for each clone.(D) Fold change of percentage of X-GFP+ cells (from SSEA1+ cells) in IFNγ-treated cells compared to untreated controls on day 7 of reprogramming, measured by flow cytometry.Bars represent the average X-GFP fold change (IFNγ vs control) for clones with the same genotype, listed in (C).Each dot represents the mean of three technical replicates for each clone.Statistics (unpaired t-tests): ns = non-significant.Error bars represent SD. (E) RT-qPCR on mRNA for Tet1, Tet2 and Tet3 expression in day 7 SSEA1+ cells (+/-IFNγ treatment day 0-5) from 3 scrambled and 3 Tet1-/-clones.Expression levels are normalized to Gapdh (2 -ΔCT ).Statistics (paired t-tests between control and treatment within clones, unpaired t-tests for comparisons between different clones): ns = non-significant; * = p<0.05.Error bars represent SD.